This invention relates to analysis of chemical substances including particulates, especially the detection of biomaterials such as DNA by mass spectrometry (MS).
Release groups in chemistry are molecular groups which function by undergoing covalent or ligand bond cleavage under certain chemical or physical conditions. They are used to link one substance to another in cases where the linked substances later need to be released. For example, in solid phase organic synthesis, a release group may be employed to link the compounds being synthesized to a solid support. In chemical analysis, a signal group may be linked by a release group to an analyte, and then the resulting signal-release group-analyte conjugate can be measured by releasing and detecting the signal group. Release groups also are used sometimes in affinity chromatography to effect the temporary attachment of a ligand to a solid support.
Field desorption mass spectrometry (FD-MS) involves the desorption of an ion from a surface by an intense electrical field (e.g. 10.sup.6 -10.sup.8 V/cm). Such field intensity is achieved by desorbing the sample from a sharp tip or edge, as provided, for example, by microscopic needles deposited pyrolytically from a carbon source onto a wire. FD-MS is a very "soft" ionization technique, but when heat is used to enhance sample desorption, fragment ions are more likely to form.
Another known analytical method is laser desorption MS (LD-MS), e.g., direct and matrix-assisted laser desorption ionization (MALDI), where only the latter employs a matrix which absorbs the energy of the laser pulse. The resulting disturbance of the matrix in turn desorbs analyte which is present within or on the surface of the matrix. In direct LD, the analyte per se (and most likely also the solid support on which it is deposited) absorbs the laser energy, leading to desorption.
DNA sequencing by MS has been studied, including MALDI and electrospray techniques employing several strategies such as measurement of dideoxy sequencing ladders, enzymatic ladder sequencing, and sequencing by gas-phase fragmentation. Practical DNA sequencing by mass spectrometry, however, has not progressed much beyond the 120-mer level, largely due to problems with depurination, fragmentation and adduct ions. This includes loss of signal strength for longer DNA fragments.
Covalent MS has been used to demonstrate that samples covalently bound to a solid surface can be measured directly by desorption mass spectrometry. For example, a covalently bound peptide on a resin particle has been detected by MALDIMS. The peptide was linked to the particle by a photolabile .alpha.-methylphenacyl ester linker, and a pulse of photons from a nitrogen laser both cleaved this group and desorbed the peptide. Similar measurement of a covalently-bound peptide, involving a photolabile benzyloxy group, has been accomplished by TOF-SIMS, and peptides have been detected by laser desorption TOF-MS where a photolabile pyridinium group was employed to covalently link the peptide to the probe surface. However, these latter two studies were conducted without applying an electrical field.
While time-of-flight mass spectrometry (TOF-MS) potentially offers high sensitivity and throughput at low cost for detection of chemical substances including macromolecules and particulates, it has been limited especially by the performance of the ion source, where the analyte in the sample is transformed into gas phase ions. If these analyte ions initially are spread out in energy or space, traveling in different directions, contaminated with nonanalyte substances (including the formation of undesirable adducts or complexes), or have energies which make them less detectable or cause any undesired fragmentation, then the overall performance of the TOF-MS technique will be poorer. There is a great need to improve the ion source in TOF-MS (and in other MS techniques such as those using ion traps for detection) in a way that overcomes these problems. For example, DNA sequencing by TOF-MS of DNA dideoxy sequencing ladders could be improved greatly, potentially making this the best way to do DNA sequencing in terms of speed, cost, accuracy and sensitivity.